The present invention is related to a method for enhancing or accelerating re-epithelialization or re-endothelialization of a tissue. Examples of re-epithelialization in which the invention is particularly suited include, but are not limited to, re-epithelialization of (a) skin following surgical wounds, (b) skin abrasions caused by mechanical trauma, caustic agents or burns, (c) cornea following cataract surgery or corneal transplants, (d) mucosal epithelium (respiratory, gastrointestinal, genitourinary, mammary, oral cavity, ocular tissue, liver and kidney) following infection, nonpathological etiologies or drug therapy, (e) skin following grafting and (f) renal tubule following acute tubular necrosis. Examples of re-endothelialization in which the invention is particularly suited include, but are not limited to, re-endothelialization (or regrowth of endothelium) in blood vessels following angioplasty, lysis of fibrin clots or lysis or mechanical disruption of thrombi in coronary arteries. The invention is especially suited for the re-epithelialization of donor sites from which epidermal tissue is harvested for application to burn sites and for repair of the tissue and microvasculature of thermally injured skin. In accordance with the present invention, the time to complete re-epithelialization or re-endothelialization is enhanced or accelerated by administering a dehydroepiandrosterone (DHEA) derivative.
The publications and other materials used herein to illuminate the background of the invention and in particular cases, to provide additional details respecting the practice, are incorporated by reference, and for convenience are nuinerically referenced in the following text and respectively grouped in the appended bibliography.
After a lesion occurs in the epidermis, it becomes critical to survival that access of the environment to the dermis is blocked without delay. In this event, the body effects wound closure in two temporally related steps: within minutes by the formation of a blood clot, which reestablishes a temporary barrier, and then within hours to days by the movement of residual epithelium below the clot and over the underlying dermis - - - the process of re-epithelialization.
The first step, involving blood clot formation and its dependence on vessel wall, platelets and coagulation proteins, is the subject of a recent review (1).
Characteristic of all epithelial cells is the propensity to cover a free surface. Clearly, in order to cover a denuded surface, epithelial cells must (1) move, and (2) grow over that wounded area. Although both processes are stimulated by wounding, the more important process in early wound closure is cell migration, which is independent of cell division (2-6). Indeed, under experimental conditions, blocking of cell division has no effect on the rate of epithelial cell movement or wound closure (7-9). The migrating cells arise from the residual epithelium at the periphery of the lesion or, more often, from the residual hair or sweat structure at the wound base. In large, deep cutaneous lesions, the epithelium that covers the wound area arises from the wound periphery. In small, superficial cutaneous wounds, however, most of the epithelium arises from the residual pilosebaceous or eccrine structure (5, 10, 11). Recent observations suggest, however, that under some circumstances mesenchymal cells may transform and become part of the regenerating epithelium (12); however, that phenomenon probably plays a minor role in the closure of most wounds.
Re-epithelialization occurs most rapidly over a superficial wound that leaves the basement membrane zone intact. In the repair of a suction blister, for example, in which the floor of the wound consists of an intact lamina densa (suction causes the separation of the epidermis from the dermis within the lamina lucida), short tongues of epithelial cells rapidly (within 12 to 24 hours) grow out from the residual epithelial structures (13). By 24 to 72 hours, most of the wound base is covered by a thin layer of epithelium, and by four days it is covered by layered keratinocytes (13, 14).
In all systems, it is the basal cell, i.e., the cell attached to the substratum, that responds to wounding and initiates migration. These marginal cells flatten out in the direction of the wound and send out cytoplasmic projections over the substratum (15, 16). In preparation for their movement, the epithelial cells loosen their intercellular and substratum attachments. They have hemidesmosomal junctions, their tonofilaments withdraw from the cell periphery, and the basement membrane zone becomes less well-defined (13,17-19). In addition, the cells at the leading edge become actively phagocytic, picking up tissue debris and erythrocytes. This phagocytic property of epidermal cells can be illustrated in the laboratory, using fluoresceincoated beads or Thorotrast particles, which are taken up by epidermal cells (16, 17, 20). This property is enhanced by the fibronectin in wound fluid (21).
Within one or two days, epithelial cells behind the migrating front begin to proliferate, generating new populations of cells to cover the wound (6, 13). Once epithelialization is complete and the wound area is covered, the epithelial cells revert to their normal prototype and reassume their intercellular and basement membrane contacts.
Re-epithelialization over any wound will occur, like an unrolling carpet or a military phalanx, by the movement of epithelial cells as a sheet. Considering the tight intercellular cohesions that epithelial cells share, it is not surprising that these cells do not migrate over a wound as single cells, but instead as small clusters or sheets. When sheets of epithelial cells have been observed directly, the cells at the margin of the moving sheet appeared to be actively motile while the cells behind (or above, in a stratified layer) the marginal cells were passively dragged along (22, 23). If attachment of the marginal cells to the substrate is disturbed, the migrating sheet, under tension, will withdraw. This mode of sheet movement, referred to as the sliding model of wound closure, has been demonstrated directly for epithelial cells in tissue culture (22), for embryonic epithelial movement (24), for amphibian wound closure (23), and for corneal wound closure (25).
It is much more difficult to study mammalian cutaneous wound closure directly because of the thickness and opacity of the dermis. Moreover, the migrating epithelial sheet of mammalian epidermis is multi-layered and thus more complex than those systems illustrating the sliding model. For the repairing mammalian epidermis, Winter (26) proposed the "leap-frog" model or epidermal sheet movement. This model was deduced indirectly from ultrastructural morphological data which suggested that cells at the migrating front adhere to the substrate only to be replaced at the front, in turn, by the cells above and behind it. Successively, then, submarginal cells are conceived to crawl over the newly adherent basal cells in a leap-frog fashion. Cell marker studies have been presented in support of this model wherein keratin antigens found in suprabasal cells of the intact epidermis (KI0, KI) are found in the basal cells of the migrating tip. Although one may ascribe these results to cell movement, these changes may also be explained by the ability of keratinocytes to switch their differentiation pattern after injury to express a keratin that normally is not found among the cells in the basal layer (28).
Although the data are indirect, the leap-frog model of mammalian epidermal wound closure has many proponents (6, 13, 27-30). As the issue is not yet resolved, it is currently reasonable to contend that simple epithelium moves by the sliding model while multilayered epithelium may manifest a more complex pattern. In mammals, either or both mechanisms (sliding and leap-frogging) may function in wound closure, depending on the state and character of the epithelium affected (31).
It is desired to identify compounds which will enhance the rate of or accelerate re-epithelialization or re-endothelialization, thus aiding in the re-epithelialization or re-endothelialization of tissue such as noted above.
DHEA is an endogenous androgenic steroid which serves as the primary precursor in the biosynthesis of both androgens and estrogens (32) and which has been shown to have a myriad of biological activities. DHEA has been reported to play a mitigating role in obesity, diabetes, carcinogenesis, autoimmunity, neurological loss of memory (33-36), and the negative effects of GCS on IL-2 production by murine T cells (37). Araneo et al. (38) has shown that the administration of DHEA to burned mice within one hour after injury resulted in the preservation of normal immunologic competence, including the normal capacity to produce T-cell-derived lymphokines, the generation of cellular immune responses and the ability to resist an induced infection. Eich et al. (39, 40) describes the use of DHEA to reduce the rate of platelet aggregation and the use of DHEA or DHEA-sulfate (DHEA-S) to reduce the production of thromboxane, respectively.
Nestler et al. (41) shows that administration of DHEA was able in human patients to reduce body fat mass, increase muscle mass, lower LDL cholesterol levels without affecting HDL cholesterol levels, lower serum apolipoprotein B levels, and not affect tissue sensitivity to insulin. Kent (42) reported DHEA to be a "miracle drug" which may prevent obesity, aging, diabetes mellitus and heart disease. DHEA was widely prescribed as a drug treatment for many years. However, the Food and Drug Administration recently restricted its use. DHEA is readily interconvertible with its sulfate ester DHEA-S through the action of intracellular sulfatases and sulfotransferases.
Daynes et al. (43) shows that administration of certain DHEA derivatives are useful for the reducing or preventing progressive tissue necrosis, reperfusion injury, bacterial translocation and adult respiratory distress syndrome. Daynes et al. (44) shows that the administration of DHEAS and other DHEA derivatives are also suitable for these uses. Finally, Araneo et al. (45) shows that DHEA derivatives are useful for reducing or preventing pulmonary hypertension. Despite the myriad of biological activities reported for DHEA derivatives, DHEA derivatives have not been reported to have any affect on re-epithelialization.